GSM7658394: VMT_HCC1599; Homo sapiens; RNA-Seq
Media was aspirated from each well and the high- throughput plates were inverted so that the device layer was facing up. The bottom membrane was gently peeled off the device to expose the tissue chambers. Devices were first washed with 500 µL of HBSS. To each device unit, 100 µL of TrypLE was added and allowed to sit as a bubble on top of the device while the plate was put back in the 37 ◦C incubator for 5 minutes. After digestion, a P200 pipet was used to pipet up and down across the tissue chambers to collect the tissues in the TrypLE. The pipet tip was moved back and forth across each device to ensure full removal of the tissue and collected into a 15 mL conical with EGM2 to neutralize the TrypLE. For full removal of remaining cells from the tissue chamber, 500 µL of EGM2 was added to each device unit and washed with the P200. Digestion solution containing the dislodged tissues was centrifuged at 300xg for 5 minutes at 4 ◦C to pellet single cells and whole tissues. Media was carefully aspirated and a solution of 1 mg/mL (200 U/mL) collagenase III in HBSS was added to the undigested tissues. After gentle pipetting, the solution was allowed to sit for 2 minutes at room temperature before pipetting gently again to dissociate the gel. Digestion mix was washed with 10 mL of EGM2 and centrifuged at 300xg for 5 minutes at 4 ◦C. Cells were resuspended into 1X DPBS with 1% human serum albumin and passed through a pre-wetted 70 µm filter by spinning at 200xg for 1 minute. Cells were counted and volume was adjusted so that total cells were at a concentration of 1000 cell per uL. Cellular suspensions were loaded onto a Chromium Single Cell Instrument (10X Genomics) to generate single-cell gel beads in emulsion (GEMs). GEMs were processed to generate cDNA libraries by using 10X Genomics v2 chemistry according to the Chromium Single Cell 3' Reagents kits v2 user guide: CG00052 Rev B. Quantification of cDNA libraries was performed using Qubit dsDNA HS Assay kit (Life Technologies Q32851), high-sensitivity DNA chips (Agilent 5067-4626) and KAPA qPCR (Kapa Biosystems KK4824). Libraries were sequenced on an Illumina NovaSeq6000 to achieve an average of 50,000 reads per cell.